Available online at www.sciencedirect.com

Nuclear Medicine and Biology 36 (2009) 403 – 409
www.elsevier.com/locate/nucmedbio

Development of microwave-based automated nucleophilic[ F]fluorination system and its application to the productionof [18 F]flumazenil
18

Katheryn S. Mandapa , Tatsuo Idoa,b , Yasushi Kiyonoa,⁎, Masato Kobayashia ,Talakad G. Lohitha , Tetsuya Moria , Shingo Kasamatsua , Takashi Kudoa ,Hidehiko Okazawaa , Yasuhisa FujibayashiaaBiomedical Imaging Research Center, University of Fukui, Fukui 910-1193, Japanb
Japan Radioisotope Association, Tokyo 113-8491, Japan
Received 24 December 2008; received in revised form 21 January 2009; accepted 23 January 2009

Abstract
Introduction: This study presents the development of an automated radiosynthesis system integrating a microwave reactor and its subsequent
application in the synthesis of [18F]flumazenil, a potentially useful compound in the evaluation of central benzodiazepine receptor density.
Methods: Preparation of dry [K/K222]+18F− complex and radiofluorination of the nitro-flumazenil precursor were achieved using thedeveloped microwave-based radiosynthesis system. The crude product was prepurified in a C18 cartridge followed by reversed-phase
preparative high-performance liquid chromatography. The isolated [18F]flumazenil was evaporated in vacuo and reconstituted in an ethanolfree solution.
Results: Optimum incorporation of 18F− in the nitro-precursor was achieved in 5 min time utilizing 2 mg of precursor in N,Ndimethylformamide reacted at 160°C which gave an incorporation yield of 40±5%. The radiochemical yield obtained at the end of synthesis
was 26±4% (EOB) with a radiochemical purity of N99% and a total synthesis time of about 55–60 min. The produced [18F]flumazenil wasobserved to be stable for at least 8 h.
Conclusion: The developed [18F]flumazenil radiosynthesis system offers shorter reaction time, simplicity in operation and applicability foruse in routine clinical practice.
© 2009 Elsevier Inc. All rights reserved.
Keywords: [18F]flumazenil; Microwave; Radiofluorination; Central benzodiazepine receptors

1. Introduction
One of the important aspects in positron emission
tomography (PET) research is to label the compound ofinterest in the least possible time. Previous reports haveshown that the utilization of microwaves poses the advantage
over conventional heating of performing the labeling reaction
in a shorter time [1–4]. The increase in reaction rate could be
attributed to the ability of the microwave to couple directlywith the sample mixture possessing polar properties [5].

⁎ Corresponding author. Tel.: +81 776 61 8420; fax: +81 776 61 8170.
E-mail address: ykiyono@u-fukui.ac.jp (Y. Kiyono).
0969-8051/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2009.01.011

Furthermore, the radiochemical yield using the microwave
method has been reported to be similar to or higher than thatof the conventional method in the synthesis of 2-[18F]fluoro2-deoxyglucose [4] and 18F-radiofluorination of selectedaromatic and aliphatic model compounds [1–3]. However,the complexity of the microwave system hinders thedevelopment of an automated radiosynthesis system, andonly results obtained by manual operation have been reported.
In this study, we developed an automatic system integrating asingle-mode microwave reactor, which provides practicableroutine production and less radiation exposure to the operator,and optimized it for the synthesis of [18F]flumazenil as well as
formulation of the ethanol-free injectable solution.
Radiolabeled flumazenil is an important radiopharmaceutical for the assessment of the central benzodiazepine

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K.S. Mandap et al. / Nuclear Medicine and Biology 36 (2009) 403–409

receptor (cBZR) concentration [6]. Its carbon-11-labeledanalog ([11C]flumazenil, Fig. 1, I) has been well studied for
cBZR evaluation in epilepsy [7], panic disorder [8],Huntington's disease [9] and identification of irreversibly
damaged tissue after acute stroke [10,11] using PET
imaging. However, due to the short half-life of carbon-11(20.4 min), usage of this radiopharmaceutical is limited tocyclotron-equipped centers. On the other hand, the fluorine18 analog, [18 F]flumazenil (Fig. 1, II), presents anadvantage with a longer half-life of 109.8 min. Moreover,a PET study conducted on the brain of a cynomolgus
monkey showed similar uptake patterns of [18F]flumazenil
and [11C]flumazenil [12]. Also, [18F]flumazenil in humanbrain showed more stable binding potential values and a lessnoisy image than [11C]flumazenil [13], thus substantiating
the potential of [18F]flumazenil as a substitute to the widelyused [11C]flumazenil.
Earlier reports on [18F]flumazenil synthesis were conducted via isotopic exchange of 19F for 18F using flumazenil(Fig. 1, III) as the precursor in a synthesis time of 45 min[14,15]. However, in isotopic exchange, specific activity
could be low due to the presence of a carrier (the precursor
flumazenil). Hence, further development on [18F]flumazenil
synthesis was reported by the same research group wherein
the utilization of nitro-flumazenil (Fig. 1, IV) as a precursor
resulted in high specific activity which is essential in brain
receptor imaging studies. However, a longer synthesis timeof about 75 min was reported [12].
This study presents an advancement in the radiosynthesisof [18F]flumazenil with the aim of improving the radiolabeling process via microwave dielectric heating. Then, using theopportunity of the shorter synthesis process, we added thesolvent exchange system to obtain ethanol-free [18 F]
flumazenil injectable solution. Ethanol is a common additive
to enhance the solubility of lipophilic drugs such as
flumazenil, but it can cause an anaphylactoid reaction withrather high incidence, especially in Asian races. Finally, asmall animal PET study in rats was performed to confirm theavailability of the new formula of [18F]flumazenil.

Fig. 1. Basic structure of flumazenil and its analogs.

2. Materials and methods
2.1. Reagents
All reagents utilized were of at least analytical grade andobtained from Sigma-Aldrich (St. Louis, MO, USA) unlessotherwise specified. Anhydrous N,N-dimethylformamide(DMF) was obtained from Kanto Chemical (Tokyo, Japan)and Aldrich (St. Louis, MO, USA). The nitro-flumazenil
precursor (ethyl5,6-dihydro-5-methyl-8-nitro-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) and standard
flumazenil (ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4Himidazo[1,5-a] [1,4]benzo-diazepine-3-carboxylate) werepurchased from Pharmasynth AS (Tartu, Estonia) andutilized as supplied. For the animal experiments, flumazenil
obtained from Sigma (St. Louis, MO, USA) was used.
Dimethyldichlorosilane (DMDCS) was purchased from
WAKO Pure Chemical Industries (Osaka, Japan), andhigh-performance liquid chromatography (HPLC)-grade
solvents were from Kanto Chemical.
2.2. Microwave reactor system
The radiosynthesis was performed using a CEM Discover(Matthews, NC, USA) single-mode microwave reactor
system. This system is capable of continuous microwave
power delivery with selectable power output (0–300 W) anda programmable temperature from 25°C to 250°C. It isequipped with an infrared temperature control system
located below the microwave cavity floor which monitorsand controls the temperature conditions of the reaction
vessel. The temperature sensor is used in a feedback loopwith the magnetron to regulate the power output which
maintains the temperature set-point. The 10-ml-thick walled(length=9 cm) Pyrex reaction vessel supplied with the
instrument was utilized for the radiolabeling reactions. The
reaction vessel was precoated prior to its use with DMDCS,
an alkyl-substituted silicon tetrahydride reagent, to minimize
the product adsorption on the inside wall.
The reaction vessel cap was modified to automate theradiosynthesis process. It was especially fabricated using
polyetheretherketone (PEEK) polymer with an aluminumbase having an access port of 1.65 cm which can exactly holdthe mouth of the 10-ml reaction vessel. The PEEK reaction
vessel cap can be tightly screwed to the aluminum base, andby using a Teflon spacer disc, it can be completely sealed,making pressurized reactions achievable. The PEEK reaction
vessel cap has access ports for inserting PEEK or Teflon
tubings of 1/16″ outer diameter which can be utilized fortransfer of reagents in and out of the reaction vessel and also
serve as inlet/outlet for Argon (Ar) gas and vacuum. To
monitor the pressure inside the reaction vessel, a pressuregauge was connected. Further developments were made byattaching a series of six-way and three-way valves to thesystem, and with the aid of a Program Logic Controller(PLC, Mitsubishi, Tokyo, Japan) and the Wizcon software(version 7.61, Elutions, France), radiosynthesis could beK.S. Mandap et al. / Nuclear Medicine and Biology 36 (2009) 403–409

controlled using a computer (Fig. 2). The developed system
is able to perform open or closed system reactions and iscapable of stop and flow operation that facilitates the transfer
of reagents in/out of the reaction vessel and the end-productfor purification.
2.3. Radiosynthesis
No carrier added 18F− was produced via 18O(p,n)18F
reaction using the RDS eclipse RD/HP cyclotron (Siemens/
CTI, Knoxville, TN, USA). The 18F− was first trapped intoa SepPak Light QMA cartridge (WATERS, Milford, MA,
USA; preconditioned with 1 ml of 0.2 M K2CO3 and
10 ml of water) followed by elution with K2CO3/K222
solution in MeCN/H2O (96:4). The K2CO3/K222 solution
utilized was based on a previous study [16] with a slight
modification wherein only 1 ml of the recommended solution was used. The eluted [K/K222]+18F− complex was thendried at a temperature of 110°C with an average microwave power of about 150 W under vacuum and Ar flow
(20 ml/min). The dried complex was used either directlyor azeotropic drying was conducted by adding 1 ml ofanhydrous acetonitrile (3×).
Nitro-flumazenil precursor (1–8 mg) dissolved in anhydrous solvent [acetonitrile, DMF or dimethyl sulfoxide

405(DMSO); (0.5–2.0 ml)] was added to the dried [K/K222]+18F− complex, and experiments were conducted(under closed system reaction) to optimize different
variables including the solvent system, labeling temperature,reaction time, nitro-precursor amount and reaction vesselpressure. The crude product was then cooled using
compressed air (option provided in the microwave system)
followed by dilution with 4.5 ml water and adsorption on a
SepPak Light C18 cartridge (WATERS). During optimization of the radiofluorination, samples from the crude productwere measured for incorporation yield using radioTLC, andthe result obtained was corrected based on the percent
distribution of the radioactivity obtained in the reaction
mixture and in the reaction vial. After washing the SepPak
Light C18 cartridge with 5 ml water, the trapped [18F]
flumazenil was then eluted with 0.7 ml of acetonitrile/water(7:3) and diluted with 0.7 ml of water prior to purification in
the HPLC system.
2.4. HPLC purification and reformulation of end-product
The HPLC system utilized for purification consisted of a
Shimadzu LC-10AD pump (Kyoto, Japan), Shimadzu
SPD-10AVP UV detector and Ludlum 44-10 NaI(Tl)radioactive detector (Ludlum Measurements, Sweetwater,

Fig. 2. Control panel screen of the developed radiosynthesis system (AI, automatic injector; SRU, solvent replacement unit).

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K.S. Mandap et al. / Nuclear Medicine and Biology 36 (2009) 403–409

TX, USA). Purification was achieved using μBondapak
C18 (7.8×300 mm) column (WATERS) with acetonitrile/water (25:75) as mobile phase at a flow rate of 3 ml/min.
The isolated [18F]flumazenil was then directly transferred
to a solvent replacement unit (JFE Holdings, Tokyo, Japan)
wherein removal of the solvent system was performed invacuo. The residue was then redissolved in 0.01% Tween
80 in normal saline solution and passed through a sterile
filter (Millipore-Millex LG, Billerica, MA, USA).
2.5. End-product evaluation
The radiochemical purity and stability of [18F]flumazenil
were determined by radioTLC performed using RP18 F254
TLC plate (Merck and Co., Whitehouse Station, NJ, USA)with ethyl acetate/ethanol (95:5) as mobile phase. Utilizing
the AR2000 TLC scanner (Bioscan, Washington, DC, USA),
the Rf value of [18F]flumazenil was determined at around0.60 and confirmed by coelution with standard flumazenil(visualized under ultraviolet light). Specific activity andradiochemical purity were determined by analytical HPLC
equipped with a Shimadzu LC-20AD pump, Shimadzu SPD20A UV spectrophotometer and Ludlum 44-10 NaI(Tl)radioactive detector and using a Cosmosil 5C18-MS-II(4.6×150 mm, Nacalai Tesque, Kyoto, Japan) column withacetonitrile/water (3:7) as mobile phase at a flow rate of
1 ml/min and a column oven temperature of 27°C. The [18F]
flumazenil peak detected at 4.70 min was verified bycoinjection with standard flumazenil.
2.6. Small animal PET imaging
All animal studies were conducted in observance of the
Guidelines of the Institutional Animal Care and Use
Committee of the Faculty of Medical Sciences at the
University of Fukui, Fukui, Japan, which conforms to theFig. 3. (A) Representative PET images of a rat from each study at 0–5, 10–15,15–20 and 30–35 min acquisition time (top: normal study, middle: displacement
study, bottom: blocking study). (B) Representative circular ROIs drawn on rat brain cortex image. (C) Time course of radioactivity in the rat brain (n=3 per study)
after injection of 18.5 MBq [18F]flumazenil. In the blocking and displacement experiments, 100 μg of cold flumazenil was injected 15 min before and after the
injection of [18F]flumazenil, respectively. Anesthesia was maintained by intermittent iv injection of chloral hydrate.

K.S. Mandap et al. / Nuclear Medicine and Biology 36 (2009) 403–409

Guide for the Care and Use of Laboratory Animals (USNational Institute of Health Publication No. 85-23, revisedin 1996). The male Sprague–Dawley (SD) rats (9–10
weeks old, 300–360 g) were obtained from Japan Slc(Shizuoka, Japan).
PET brain imaging on SD rats (n=3 per study) was
performed using a dedicated small animal PET scanner(Hamamatsu SHR-41000). The scanner has a transaxial
resolution of 2.0 mm FWHM in the center of the field of view
and an axial resolution of about 2.8 mm FWHM on the axis of
the ring [17]. The SD rats (300–360 g) were first anesthetizedby intraperitoneal injection of chloral hydrate (400 mg/kg
body weight) and then maintained by intermittent intravenous (iv) injection. Transmission scan was then acquired for
60 min with an axial field of view of 100 mm covering thehead and proximal part of the trunk. This was followed by iv
bolus injection of about 18.5 MBq of [18F]flumazenil.
Dynamic emission scan was then acquired for 60 min andthe coincidence data were stored in list mode. The list mode
data were edited to a 3D data set and then converted to 213
slices of 2D sinograms by Fourier rebinning algorithm.
Reconstruction of 2D sinograms was performed using astandard 2D filtered back projection algorithm at selected
time frames. The blocking or displacement studies were
performed by iv injection of 100 μg of cold flumazenil 15 min
before or after [18F]flumazenil administration, respectively.
Circular regions of interest (ROIs) were drawn on the rat
brain cortex (Fig. 3B) for all the time frames and their mean
counts were obtained. The counts per pixel per minute fromthe ROIs were converted to tracer activity (Bq/ml) using across-calibration factor obtained by scanning a cylindricalphantom with known 18F activity concentration. Assuming atissue density of 1 g/ml, tracer activity was divided by the407

injected dose (Bq) to obtain an image ROI-derived tissue
uptake index in % ID/g.
3. Results and discussion
3.1. Radiosynthesis with microwave reactor system
The utilization of microwave for radiopharmaceuticalsynthesis is one of the techniques that can be used to reduceradiolabeling time. However, to date, a radiosynthesis
system that integrates microwave dielectric heating is stilllacking for widespread use. In this study, a radiosynthesis
system incorporating a microwave reactor was developedand employed for the synthesis of [18F]flumazenil. The
developed system can be remotely operated which thenminimizes the operator's exposure to radiation, simplifies theradiopharmaceutical production and facilitate the optimization of the radiosynthesis procedure in a closed environment.
The microwave reactor setup was initially utilized for the
preparation of dry [K/K222]+18F− complex. One milliliter ofthe prepared K2CO3/K222 solution was enough to elute the[K/K222]+18 F− complex from the SepPak Light QMA
cartridge with N96% elution yield. Dry [K/K222]+18 F−
complex was obtained at a temperature of 110°C with anaverage microwave power of 150 W for a period of 110 s.
Moreover, no significant difference in [18F]flumazenil yield
was observed for runs conducted with or without additionalazeotropic drying (Table 1, Series 6 and 7) which is inaccordance with the previous report using other modelcompounds [16]. Dry [K/K222]+18F− complex could thus beprepared in less than 2 min.
In order to optimize the 18F− incorporation rate into thenitro-flumazenil precursor, the solvent system, nitro-

Table 1
Summary of [18F]flumazenil labeling parameters optimization
Series no.

NFMZ (mg)

Molar ratio NFMZ/[K222/K2CO3]

Reaction
temperature (°C)

Solvent

Pressure (kPa)/
time (min)No. of runs%[18F]FMZ yield(RTLC)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

2
2
5
5
2
2
2
1
4
5
8
2
2
2
2
2
2
2
2

0.5:1
0.5:1
1.25:1
1.25:1
0.5:1
0.5:1
0.5:1
0.25:1
1:1
1.25:1
2:1
0.5:1
0.5:1
0.5:1
0.5:1
0.5:1
0.5:1
0.5:1
0.5:1

90
160
160
200
160
160
160
160
160
160
160
140
180
200
160
160
160
160
160

Acetonitrile
Acetonitrile
Acetonitrile
DMSO
DMSO
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF

0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
100/10
100/5
200/5
200/2
200/10

1
1
1
1
2
3
7
1
3
3
3
3
3
3
1
3
7
3
3

b1
8
9
27
25±1
31±4a
30±6
b3
27±6
30±8
29±9
15±2
21±4
14±6
39
33±3
40±5
21±2
34±6

a

With additional azeotropic drying.

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K.S. Mandap et al. / Nuclear Medicine and Biology 36 (2009) 403–409

flumazenil precursor amount, labeling temperature, labelingtime and reaction vessel pressure were varied (Table 1). For
solvent system optimization, three commonly used solvent
systems for radiolabeling reaction were tested, that is,acetonitrile, DMF and DMSO. These solvents have medium
to high dielectric loss and hence are likely to couple
efficiently with the microwave energy leading to rapid
heating [5]. Radiolabeling with acetonitrile resulted in a poor
incorporation yield while DMSO resulted in a better yieldthan acetonitrile but was slightly less compared to DMF atequivalent conditions (Table 1, Series 1–7). Furthermore, thereaction with DMSO showed several 18F-labeled by-products
besides [18F]flumazenil as determined by the HPLC of thecrude reaction mixture while the DMF reaction gave only one
minor 18F-labeled by-product besides [18F]flumazenil. DMF
was then determined as the most appropriate solvent due to its
higher yield and lesser 18F-labeled by-product.
The incorporation yield by varying the amount of nitroflumazenil keeping the K2CO3/K222 constant showed nosignificant increase in incorporation yield from 2 mg ofnitro-flumazenil up to 8 mg, while 1 mg of nitro-flumazenil(nitro-flumazenil/[K2CO3/K222] ratio of 0.25:1) gave lessthan 3% yield (Table 1, Series 7–11) which was perhaps dueto the degradation of the produced [18F]flumazenil in a morebasic environment [14,15]. In this study, a nitro-flumazenil/[K2CO3/K222] ratio of 0.5:1 was enough to obtain the
optimum yield as compared to the 1:1 ratio recommended in
the previous study [12]. This result shows that a minimal
amount of nitro-flumazenil precursor was required for theradiosynthesis, thereby saving on the production cost andfacilitating the purification process (Table 2). The reductionin the amount of precursor is one of the special features
observed in microwave reactions [3].
Utilizing DMF as a solvent, the optimum labelingtemperature was determined to be at 160°C with an average
microwave power of around 30 W (Table 1, Series 7, 12–
Table 2Comparison of [18F]flumazenil radiosynthesis parameters and end-productspecification obtained in this study with those in the literature
Properties

Precursor
Precursor amount (mg)[K/K222]+18F−
complex drying
time (min)Radiofluorination
time (min)HPLC purification
time (min)RCY (EOB)RCY (EOS)
RCP
Specific activity(GBq/μmol)
Total synthesistime (min)Conductive heating

Microwave

Ref. [12]Ref. [15]This study

Nitro-flumazenil
8–

Flumazenil
2–

Nitro-flumazenil
2
2

30

15

5

22–24

–

17–19

30%
19%
–
185

47%
N97%
0.37

26±4%
18±3%
N99%
295±54

75–80

45

55–60

14). The lower yield observed at higher temperatures may be
attributed to degradation of [18F]flumazenil [15]. Furthermore, a better yield with a shorter reaction time (5 min) was
observed as a result of pressurizing the reaction vessel with200 kPa of Ar gas prior to labeling (Table 1, Series 7, 15–19). Utilizing 2 mg of precursor in DMF reacted at 160°C(with 200 kPa reaction vessel pressure) and employing an
average microwave power of 30 W gave an incorporation
yield of 40±5% in 5 min time.
The crude product was then diluted with water and prepurified in a SepPak Light C18 cartridge to remove the DMF
solvent prior to HPLC purification. [18F]Flumazenil wasthen isolated between 17 and 19 min. Reformulation of the
final product in ethanol/saline solution can be achieved with
the SepPak C18 cartridge, which is convenient and requires
less time. However, this might present untoward effects onpatients with alcohol intolerance, so the isolated [18F]
flumazenil was subjected to evaporation in order to remove
the HPLC solvent and was then redissolved in 0.01% Tween
80, a surfactant approved for use in parenteral products [18],in normal saline solution. The radiochemical yield obtainedat the end of synthesis was 26±4% (EOB) with a radiochemical purity of N99% and a total synthesis time of about
55–60 min (Table 2). The specific activity was recorded ataround 295 GBq/μmol (EOS). Furthermore, the obtained[18F]flumazenil was observed to be stable for at least 8 h.
The developed radiosynthesis system which uses microwave dielectric heating is robust, flexible and was easily
manipulated to produce [18F]flumazenil. In Table 2, theresults of [18F]flumazenil radiosynthesis obtained in thisstudy based on microwave dielectric heating are compared to
the results of previously reported syntheses based on
conductive heating. With the microwave heating system,
the total synthesis time including improved injectable
formulation was shortened to 55–60 min as compared tothe conductive heating system which took 75–80 min using
the nitro-flumazenil as the precursor. The nitro-group to 18F
exchange reaction increased the specific activity to about
185 GBq/μmol [12] in contrast to 0.37 GBq/μmol reported
for the isotopic exchange reaction [15]. In this study, it wasnoted from the HPLC chromatogram of the [18F]flumazenil
product (data not shown) that the cold flumazenil which wassynthesized by F-19 from Teflon tubes and cyclotron lines,
and/or nonlabeled organic impurity which corresponds to the
UV absorption of flumazenil tend to be lower with smaller
amounts of the nitro-precursor. Further tests will however be
conducted to clarify this observation. The conditions
involved in this radiosynthesis (taking care to reduce
possible contamination with fluoride) along with theminimal amount of nitro-precursor gave a calculated highspecific activity of 295 GBq/μmol. Although the radiochemical yield at EOB was observed to be slightly low, thiswas compensated for by the shorter production time leading
to a comparable radiochemical yield at EOS. Using thedeveloped radiosynthesis system that incorporates a microwave reactor, the [18F]flumazenil can thus be produced at a

K.S. Mandap et al. / Nuclear Medicine and Biology 36 (2009) 403–409

shorter time, with reproducible yield, higher specific activity
and at a sufficient amount for use in PET receptor imaging
studies. Furthermore, it is simple to operate and hence waseasily modified for radiofluorination of other modelcompounds (to be published elsewhere).
3.2. Small animal PET imaging
Using dynamic PET imaging, the initial high uptake andrapid decline in retention of [18F]flumazenil in the brain was
clearly visualized (Fig. 3C). The time course of radioactivity
is similar to the results of [3H]flumazenil in vivo binding in
the mouse brain [19]. After injection of [18F]flumazenil,
there was a rapid and high level of accumulation ofradioactivity in the brains of normal rats. The high uptake
in the cortical region was observed at 3–15 min after
injection followed by quick elimination over 60 min (Fig.
3C). Fig. 3A shows representative PET images of rats.
Pretreatment with cold flumazenil suppressed the accumulation of [18F]flumazenil by around 75% (at 20 min acquisitiontime) as compared with normal rat study. Furthermore, in thedisplacement study, a marked decrease in brain radioactivityof about 80% (at 20 min acquisition time) as compared to thenormal rat study was observed 5 min after injection of thecold flumazenil. The in vivo blocking and displacement
studies performed further confirmed the specific binding of[18F]flumazenil. Gathering these data, similar bioavailability
and characteristics of the ethanol-free [18 F]flumazenil
injectable solution with those of radiolabeled flumazenil
could be visualized.
4. Conclusion
Utilizing the developed radiosynthesis system, [18F]
flumazenil was produced with ease in a minimal time andwith higher specific activity. The capability of the ethanolfree [18F]flumazenil injectable solution to clearly visualize
the cBZR distribution in rodents makes it a potential
radiopharmaceutical for molecular imaging experiments.
Several other evaluations are being conducted in order tofully establish the synthesized [18F]flumazenil's suitability
for clinical application.
The system presented is a first online microwave-based
radiosynthesis system which, in contrast to a batch system,
minimizes possible contamination from outside environment
and facilitates transfer of reagent/product. Furthermore, thesystem is capable of carrying out reactions in a completely
sealed condition, which then enables it to perform chemical
reactions requiring high pressure. The developed system alsooffers simplicity in operation and applicability for use inradiopharmaceutical production.
Acknowledgments
The authors thank Mr. Toshinao Nakakoji of CMI for
operating the cyclotron. This study was supported by the409

21st Century COE program “Biomedical Imaging Technology Integration Program” from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) Japan.
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